The purpose of this project is to investigate cholesterol esterification in the small intestine. This will be accomplished in two parts. The first part will study the regulation of intestinal mucosal acylcoenzyme A: cholesterol acyltransferase (ACAT) in vivo and in vitro. To study the regulation of ACAT by luminal factors, rabbits will be fed various fats and sterols which will alter cholesterol absorption from the gut and change serum cholesterol levels. The response of intestinal microsomal ACAT to these modifications will then be measured. ACAT activity in intestinal biopsies of Thiry-Vella fistula-rabbits fed either normal rabbit chow or a 1% cholesterol diet, will determine regulation of ACAT by serum factors. Intestinal organ culture will be used to observe changes in ACAT activity in response to various fatty acids, sterols, and hormones dependent of serum or luminal factors. The second part will challenge the accepted hypothesis that pancreatic cholesterol ester hydrolase (CEH) is the enzyme responsible for cholesterol esterification in the intestine. CEH activity will be measured in intestinal lumen, glycocalyx, and cytosol to determine if the enzyme is truly intracellular. Next, CEH will be measured in vitro under more physiological conditions than that described for the assay in the literature. Finally, the regulation of CEH will be studied by in vivo feeding experiments and in vitro organ culture, similarly to the experiments described for ACAT above. Conclusions will be made regarding the contributions of these two enzymes on intestinal cholesterol esterification. A clearer understanding of this reaction will provide much needed information on the relationship of cholesterol esterification activity in the gut to cholesterol absorption and lipoprotein composition.